Ruth Krasnow

Asthma Discordance in Twins Is Linked to Epigenetic Modifications of T Cells

T cells mediate the inflammatory responses observed in asthma among genetically susceptible individuals and have been suspected to be prone to epigenetic regulation. However, these relationships are not well established from past clinical studies that have had limited capacity to control for the effects of variable genetic predisposition and early environmental exposures. Relying on a cohort of monozygotic twins discordant for asthma we sought to determine if epigenetic modifications in T cells were associated with current asthma and explored whether such modifications were associated with second hand smoke exposures. Our study was conducted in a monozygotic twin cohort of adult twin pairs (n = 21) all discordant for asthma. Regulatory T cell (Treg) and effector T cell (Teff) subsets were assessed for levels of cellular function, protein expression, gene expression and CpG methylation within Forkhead box P3 (FOXP3) and interferon gamma-γ (IFNγ) loci. Comparisons by asthma and current report of exposure to second hand smoke were made. Treg from asthmatic discordant twins demonstrated decreased FOXP3 protein expression and impaired Treg function that was associated with increased levels of CpG methylation within the FOXP3 locus when compared to their non-asthmatic twin partner. In parallel, Teff from discordant asthmatic twins demonstrated increased methylation of the IFNγ locus, decreased IFNγ expression and reduced Teff function when compared to Teff from the non-asthmatic twin. Finally, report of current exposure to second hand smoke was associated with modifications in both Treg and Teff at the transcriptional level among asthmatics. The results of the current study provide evidence for differential function of T cell subsets in monozygotic twins discordant for asthma that are regulated by changes in DNA methylation. Our preliminary data suggest exposure to second hand smoke may augment the modified T cell responses associated with asthma.

Nicotinic Acetylcholine Receptor Variation and Response to Smoking Cessation Therapies

Objective To evaluate the association of nicotinic acetylcholine receptor (nAChR) single nucleotide polymorphism (SNP) with 7-day point prevalence abstinence (abstinence) in randomized clinical trials of smoking cessation therapies in individuals grouped by pharmacotherapy randomization to inform the development of personalized smoking cessation therapy. Materials and methods We quantified association of four SNPs at three nAChRs with abstinence in eight randomized clinical trials. Participants were 2633 outpatient treatment-seeking, self-identified European ancestry individuals smoking at least 10 cigarettes/day, recruited through advertisement, prescribed pharmacotherapy, and provided with behavioral therapy. Interventions included nicotine replacement therapy (NRT), bupropion, varenicline, placebo (PLA), or combined NRT and bupropion, and five modes of group and individual behavioral therapy. Outcome measures tested in multivariate logistic regression were end of treatment and 6 month (6MO) abstinence, with demographic, behavioral, and genetic covariates. Results ‘Risk’ alleles previously associated with smoking heaviness were significantly (P<0.05) associated with reduced abstinence in the PLA pharmacotherapy group (PG) at 6MO [for rs588765, odds ratio (95% confidence interval) 0.41 (0.17–0.99)], and at end of treatment and at 6MO [for rs1051730, 0.42 (0.19–0.93) and 0.31 (0.12–0.80)], and with increased abstinence in the NRT PG at 6MO [for rs588765, 2.07 (1.11–3.87) and for rs1051730, 2.54 (1.29–4.99)]. We observed significant heterogeneity in rs1051730 effects (F=2.48, P=0.021) between PGs. Conclusion chr15q25.1 nAChR SNP risk alleles for smoking heaviness significantly increase relapse with PLA treatment and significantly increase abstinence with NRT. These SNP–PG associations require replication in independent samples for validation, and testing in larger sample sizes to evaluate whether similar effects occur in other PGs.

Reliability of Adult Retrospective Recall of Lifetime Tobacco Use

Retrospective assessment of tobacco use underlies much of the data collected in epidemiological and genetic epidemiological research. Although individuals are asked to report lifetime tobacco use for periods spanning months to decades, the test-retest reliability intervals of the instruments often span only a few weeks to several months. The present analyses examined the test-retest reliability of retrospective tobacco use measures, including details of first use, circumstances of first use, and initial subjective reactions. The questions were part of the Lifetime Tobacco Use Questionnaire (LTUQ), a Web-based questionnaire designed to assess use of most forms of tobacco or nicotine retrospectively across the lifespan. A convenience sample of 236 men and women with history of tobacco use (Time 1 mean age, 44.9 years; 74.2% females; 75.1% regular monthly tobacco use) responded verifiably to invitations to self-administer the LTUQ two times, 2 years apart. Test-retest reliability analyses reflected high reliability for salient tobacco-use questions. Acceptable levels of reliability were observed for initial subjective reactions to smoking, if the scaled response options were dichotomized. Few differences in the reliability of recall were apparent between sexes and between age groups. These results indicate that recall of important tobacco use information can form a reliable basis for research.

Resequencing of nicotinic acetylcholine receptor genes and association of common and rare variants with the Fagerström test for nicotine dependence.

Common single-nucleotide polymorphisms (SNPs) at nicotinic acetylcholine receptor (nAChR) subunit genes have previously been associated with measures of nicotine dependence. We investigated the contribution of common SNPs and rare single-nucleotide variants (SNVs) in nAChR genes to Fagerström test for nicotine dependence (FTND) scores in treatment-seeking smokers. Exons of 10 genes were resequenced with next-generation sequencing technology in 448 European-American participants of a smoking cessation trial, and CHRNB2 and CHRNA4 were resequenced by Sanger technology to improve sequence coverage. A total of 214 SNP/SNVs were identified, of which 19.2% were excluded from analyses because of reduced completion rate, 73.9% had minor allele frequencies <5%, and 48.1% were novel relative to dbSNP build 129. We tested associations of 173 SNP/SNVs with the FTND score using data obtained from 430 individuals (18 were excluded because of reduced completion rate) using linear regression for common, the cohort allelic sum test and the weighted sum statistic for rare, and the multivariate distance matrix regression method for both common and rare SNP/SNVs. Association testing with common SNPs with adjustment for correlated tests within each gene identified a significant association with two CHRNB2 SNPs, eg, the minor allele of rs2072660 increased the mean FTND score by 0.6 Units (P=0.01). We observed a significant evidence for association with the FTND score of common and rare SNP/SNVs at CHRNA5 and CHRNB2, and of rare SNVs at CHRNA4. Both common and/or rare SNP/SNVs from multiple nAChR subunit genes are associated with the FTND score in this sample of treatment-seeking smokers.

Identification of Novel CYP2A6*1B Variants : The CYP2A6*1B Allele is Associated With Faster In Vivo Nicotine Metabolism

Cytochrome P450 2A6 (CYP2A6) is the human enzyme responsible for the majority of nicotines metabolism. CYP2A6 genetic variants contribute to the interindividual and interethnic variation in nicotine metabolism. We examined the association between the CYP2A6*1B variant and nicotines in vivo metabolism. Intravenous infusions of deuterium‐labeled nicotine were administered to 292 volunteers, 163 of whom were White and did not have common CYP2A6 variants, other than CYP2A6*1B. We discovered three novel CYP2A6*1B variants in the 3′‐flanking region of the gene that can confound genotyping assays. We found significant differences between CYP2A6*1A/*1A, CYP2A6*1A/*1B, and CYP2A6*1B/*1B groups in total nicotine clearance (17.2±5.2, 19.0±6.4, and 20.4±5.9, P<0.02), non‐renal nicotine clearance (16.4±5.0, 18.5±6.2, and 19.8±5.7, P<0.01), and the plasma trans‐3′‐hydroxycotinine/cotinine ratio (0.26±0.1, 0.26±0.1, and 0.34±0.1, P<0.001). There were also differences in total nicotine (29.4±12.9, 25.8±0.12.9, and 22.4±12.4, P<0.01), cotinine (29.2±8.1, 32.2±9.1, and 33.0±6.6, P<0.01) and trans‐3′‐hydroxycotinine (32.4±9.1, 34.2±12.3, and 41.3±11.3, P<0.001) excreted in the urine. We report evidence that CYP2A6*1B genotype is associated with faster nicotine clearance in vivo, which will be important to future CYP2A6 genotype association studies.

Association of sex steroid hormones with brain morphology and cognition in healthy elderly men

Background: There is inconsistent evidence of the presence and direction of the relationship between sex hormone concentrations and cognitive function in older men, and there is little published literature on the relationship of sex hormone concentrations and brain volume as measured by MRI. Objective: To examine the hypothesis that midlife total serum concentrations of testosterone (T), estradiol, estrone, and sex hormone binding globulin (SHBG) predict cognitive task performance and regional brain volumes at 10- to 16-year follow-up, in a longitudinal sample of World War II veteran twin men. Methods: Treating twins as individuals, linear regression models were used, adjusting analyses for age, education, depressive symptomatology, blood pressure, alcohol consumption, years of cigarette smoking, and APOE ε4 allele status. Results: There were no significant associations between sex hormone or SHBG concentrations and performance on a series of cognitive tasks measuring global and executive function, visual and verbal learning and memory. Higher midlife T concentrations were associated with larger hemispheric, frontal, and parietal regional brain volumes and with smaller left occipital brain volume. Higher estradiol and estrone concentrations were also associated with smaller right (estradiol) and both right and left (estrone) occipital volumes, but with no other brain regions. Owing to the multiple comparisons conducted, some significant associations may have occurred by chance. Conclusions: Overall, the pattern of results suggests a role for sex hormones in brain volume that predates potentially observable associations between sex hormones and cognitive task performance.

Dopamine genes and nicotine dependence in treatment seeking and community smokers

We utilized a cohort of 828 treatment-seeking self-identified white cigarette smokers (50% female) to rank candidate gene single nucleotide polymorphisms (SNPs) associated with the Fagerström Test for Nicotine Dependence (FTND), a measure of nicotine dependence which assesses quantity of cigarettes smoked and time- and place-dependent characteristics of the respondents smoking behavior. A total of 1123 SNPs at 55 autosomal candidate genes, nicotinic acetylcholine receptors and genes involved in dopaminergic function, were tested for association to baseline FTND scores adjusted for age, depression, education, sex, and study site. SNP P-values were adjusted for the number of transmission models, the number of SNPs tested per candidate gene, and their intragenic correlation. DRD2, SLC6A3, and NR4A2 SNPs with adjusted P-values <0.10 were considered sufficiently noteworthy to justify further genetic, bioinformatic, and literature analyses. Each independent signal among the top-ranked SNPs accounted for ∼1% of the FTND variance in this sample. The DRD2 SNP appears to represent a novel association with nicotine dependence. The SLC6A3 SNPs have previously been shown to be associated with SLC6A3 transcription or dopamine transporter density in vitro, in vivo, and ex vivo. Analysis of SLC6A3 and NR4A2 SNPs identified a statistically significant gene–gene interaction (P=0.001), consistent with in vitro evidence that the NR4A2 protein product (NURR1) regulates SLC6A3 transcription. A community cohort of N=175 multiplex ever-smoking pedigrees (N=423 ever smokers) provided nominal evidence for association with the FTND at these top ranked SNPs, uncorrected for multiple comparisons.

Nature Versus Nurture in Gout: A Twin Study

BACKGROUND Gouty arthritis (gout) is the most common inflammatory arthritis in the United States and several other countries. Some rare forms of gout have a known genetic basis, but the relative importance of genetic factors on the risk for the lifetime prevalence of gout is not clear. METHODS We performed a heritability analysis for hyperuricemia and gout among 514 unselected, all-male twin pairs who were a part of the National Heart, Lung, and Blood Institute twin study, a prospective observational cohort study. Statistical analyses were performed using structural equation models and maximum likelihood methods. The covariates used for adjustment in the structural equation models were identified using bivariate logistic regressions. RESULTS The study population included 253 monozygotic (MZ) and 261 dizygotic (DZ) twin pairs, aged 48 (±3) years at baseline and followed for a mean of 34 years. The lifetime prevalence of gout did not differ between MZ and DZ twins. The concordance of hyperuricemia was 53% in MZ and 24% in DZ twin pairs (P<.001). Models that quantified the relative contribution of genetic and environmental factors on phenotypic variance showed that individual variability in gout was substantially influenced by environmental factors shared between co-twins and not by genetic factors. In contrast, individual differences in hyperuricemia were influenced significantly by genetic factors. CONCLUSION Hyperuricemia is a genetic trait. Outside the context of rare genetic disorders, risk for gout is determined by the environment. This has implications for prevention and treatment approaches.

Pharmacogenetics of nicotine metabolism in twins: methods and procedures.

This article describes a pharmacogenetic investigation of nicotine metabolism in twins. One hundred and thirty-nine twin pairs (110 monozygotic and 29 dizygotic) were recruited and assessed for smoking status, zygosity, and health conditions known or suspected to affect drug metabolism. Participants underwent a 30-minute infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, followed by an 8-hour in-hospital stay. Blood and urine samples were taken at regular intervals for analysis of nicotine, cotinine, and metabolites by gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry and subsequent characterization of pharmacokinetic phenotypes. DNA was genotyped to confirm zygosity and for variation in the primary gene involved in nicotine metabolism, CYP2A6. Univariate and multivariate biometric analyses planned for the future will determine genetic and environmental influences on each pharmacokinetic measure individually and in combination with each other, and in the presence and absence of covariates, including measured genotype. When the analyses are completed, this study will result in a more complete characterization of the impact of genetic and environmental influences on nicotine and cotinine metabolic pathways than has heretofore been reported. The approach taken, with its use of a quantitative model of nicotine metabolism, highly refined metabolic phenotypes, measured genotype, and advanced tools for biometric genetic analysis, provides a model for the use of twins in next-generation studies of complex drug-metabolism phenotypes.

Test-Retest Reliability of Web-Based Retrospective Self-Report of Tobacco Exposure and Risk

Background Retrospectively collected data about the development and maintenance of behaviors that impact health are a valuable source of information. Establishing the reliability of retrospective measures is a necessary step in determining the utility of that methodology and in studying behaviors in the context of risk and protective factors. Objective The goal of this study was to examine the reliability of self-report of a specific health-affecting behavior, tobacco use, and its associated risk and protective factors as examined with a Web-based questionnaire. Methods Core tobacco use and risk behavior questions in the Lifetime Tobacco Use Questionnaire—a closed, invitation-only, password-controlled, Web-based instrument—were administered at a 2-month test-retest interval to a convenience sample of 1229 respondents aged 18 to 78 years. Tobacco use items, which covered cigarettes, cigars, smokeless tobacco, and pipe tobacco, included frequency of use, amount used, first use, and a pack-years calculation. Risk-related questions included family history of tobacco use, secondhand smoke exposure, alcohol use, and religiosity. Results Analyses of test-retest reliability indicated modest (.30 to .49), moderate (.50 to .69), or high (.70 to 1.00) reliability across nearly all questions, with minimal reliability differences in analyses by sex, age, and income grouping. Most measures of tobacco use history showed moderate to high reliability, particularly for age of first use, age of first weekly and first daily smoking, and age at first or only quit attempt. Some measures of family tobacco use history, secondhand smoke exposure, alcohol use, and religiosity also had high test-retest reliability. Reliability was modest for subjective response to first use. Conclusions The findings reflect the stability of retrospective recall of tobacco use and risk factor self-report responses in a Web-questionnaire context. Questions that are designed and tested with psychometric scrutiny can yield reliable results in a Web setting.